Abstract
In the protein disulfide-introducing system of Escherichia coli, plasma membrane-integrated DsbB oxidizes periplasmic DsbA, the primary disulfide donor. Whereas the DsbA-DsbB system utilizes the oxidizing power of ubiquinone (UQ) under aerobic conditions, menaquinone (MK) is believed to function as an immediate electron acceptor under anaerobic conditions. Here, we characterized MK reactivities with DsbB. In the absence of UQ, DsbB was complexed with MK8 in the cell. In vitro studies showed that, by binding to DsbB in a manner competitive with UQ, MK specifically oxidized Cys41 and Cys44 of DsbB and activated its catalytic function to oxidize reduced DsbA. In contrast, menadione used in earlier studies proved to be a more nonspecific oxidant of DsbB. During catalysis, MK8 underwent a spectroscopic transition to develop a visible violet color (lambdamax = 550 nm), which required a reduced state of Cys44 as shown previously for UQ color development (lambdamax = 500 nm) on DsbB. In an in vitro reaction system of MK8-dependent oxidation of DsbA at 30 degrees C, two reaction components were observed, one completing within minutes and the other taking >1 h. Both of these reaction modes were accompanied by the transition state of MK, for which the slower reaction proceeded through the disulfide-linked DsbA-DsbB(MK) intermediate. The MK-dependent pathway provides opportunities to further dissect the quinone-dependent DsbA-DsbB redox reactions.
Highlights
Disulfide bond formation, an important maturation process for envelope and secreted proteins, depends on dedicated cellular factors (Dsb proteins) (1, 2)
As a more thorough introduction and discussion of the modes of DsbB function in the oxidation of DsbA, including the current controversies, has been presented in our previous study (11), we recapitulate some salient features of UQ-DsbB interaction here
We have shown that UQ undergoes a spectroscopic transition to exhibit an absorption peak at ϳ500 nm only when it is on DsbB in certain forms (11)
Summary
Disulfide bond formation, an important maturation process for envelope and secreted proteins, depends on dedicated cellular factors (Dsb proteins) (1, 2). We have shown that UQ undergoes a spectroscopic transition to exhibit an absorption peak at ϳ500 nm only when it is on DsbB in certain forms (11). DsbA itself is not directly required for the UQ anomaly, a disulfide rearrangement in the DsbA-DsbB complex, as demonstrated by Kadokura and Beckwith (19), will generate reduced Cys[44] and the spectroscopic transition of UQ. Menaquinone-dependent Disulfide Bond Formation in E. coli posed that the spectroscopic transition of UQ represents its activated state that drives electron transfer and resolution of the DsbA-DsbB complex in a manner overcoming the reversed redox potential difference The first indication for the in vivo importance of MK was provided by our observation that mutational impairment of both the UQ and MK biosynthetic pathways results in dysfunction of the DsbA-DsbB system (9). Kadokura et al (16) showed that some Arg[48] substitutions in DsbB result in a low activity enzyme that can no longer utilize the MK analog menadione as an in vitro electronaccepting substrate
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